*Target (protein/gene name): Beta lactamase class A *NCBI Gene # or RefSeq#: 1280 [NCBI] *Protein ID (NP or XP #) or Wolbachia#: NP_878023.1 *Organism (including strain): staphylococcus aureus PC1 Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up): Staphylococcus aureus is a gram-positive bacteria grouped with Bacillus sp based on ribosomal RNA sequences. This coccus bacterium grows in both aerobic and anaerobic conditions, forming grape-like clusters. Its mainly resides in nasal membranes and skin of warm-blooded animals, causing infections ranging from mild, such as skin infections and food poisoning, to life-threatening, such as pneumonia, sepsis, osteomyelitis, and infectious endocarditis.1 The organism is highly resistant to antibiotics, including meticillin and the most potent β-lactams. Meticillin-resistant S aureus (MRSA) is the main cause of nosocomial infection worldwide and especially prominent in hospital. Beta-lactamase is involved in bacterial resistance to beta-lactam antibiotics and if a drug can inhibit this enzyme, then maybe the antiibiotics can work.
The essentiality is pretty high in MRSA infection because beta-lactamase is behind the resistance for beta-lactam antibiotics
Complex of proteins?: No - but it is in the group 2a of beta-lactamase proteins Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Yes, existing drugs are listed here: http://database.idrb.cqu.edu.cn/TTD/ZFTTDDetail.asp?ID=TTDS00228
It is druggable – similar to PBP4 target and has some known drugs already
No significant similarity between beta-lactamase and human proteins found. Blasted twice to make sure.
Current Inhibitors: This target has a lot of compounds but none of the source organisms are staphylococcus aureus and checking the targets of staphylococcus aureus, 4 main targets are listed, and this is not one them – indicating this is a pretty unstudied target in this organism. - http://www.bindingdb.org/bind/BySourceOrganism.jsp Boronic acids are active-site inhibitors of beta lactamases.
Expression Information (has it been expressed in bacterial cells): Yes, its expression system is E.coli
Purification Method: Sequential cation exchange and affinity chromatography using a phenylboronic acid-agarose affinity column to purify beta lactamase from various bacterial species.
Image of protein (PyMol with features delineated and shown separately): Dashed lines represent hydrogen bonds *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNTKFLGKTLVASALVLTTLGTGLHSSYLGLDTNKVVKTAKAEEKMTDGQLWKKVKDSLH
DSDIILSNEYETINVTYLLSNGYSSSVSAPGNDDGGHLTQSIDFKGLKQIDLTKENVYDD
FNKKLDAKNTWNSLTEKLKGLGLLQNGQKVSIYSSDSSSPVSGKVGEGVTSGGENTLTKR
FINKITID *length of your protein in Amino Acids: 188 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20.345 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 19940 M^-1 cm^-1
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): TTA AAA TTC TTA CAC TAC ACA TTA GGA GGT TTC ACT TAC CAG TTA ATC ATA TGG TTG ATC ACG TTT ATA GTA TTC ATA AGA AAG AAC GAC TGA AAC AAT AGA TTCCGA TTA GCT TTG CGA ATT AAC AAA TCA AAT ATC ATT AGT CGA AGT ATA TGT TAT TGC TTG ACG ACG TTT ATT AGT AAT TTA ATA GTC TTT TGG AAT ACT GTT TTTAAC TAA TCT ATC AGT GCA TTC ATA ATT TGA CAT TGA ATT AAA TAA GAA TTT TTT GTC TTC TTC GCT TAA TTT AGT ATC TGT GAT AAG TTT ATC TGA AGT TTA ACTGAA AGT TCA CGT TGC TAA TCA ATT TTT TTC GCT TTA TGG TAA ATC GTC ATC TGA TTT TAT TTA ATA AGT AAC TGG ATC TGT TAC TTT ATT TCT TAA TTC TTC TGAACG TTA TTT AAT TTT TTC GAT TCA ACT GAT TTC TTT TAC AAC TTT ATT GTT TGT TGC ATA ATC ACA ATC TGC CAC TGA AGT TTA AAT AAA TTC TTT TGA AGT GATATC TTT TCA TAC ATC TTC TTC TGA AAT CGA CGA ATA TCT AAC TAT ATA ATT TTT GTT AAT ATG TAT TTC TTT ATA TAA TTA ATC ATT CGT TAC TTG TTT TGA CAGAAC TCT ACA ATT TAC CGT TTT TAA TCT CGT AGT ATA GGC AAA AGT TTG ATT TAA ATA AAA TTT TAC TTC TTA ACT ACT TTC TCT ATA TAA TCA ATC AAT ACA AATATG TCA ATT ATA TTC TTC TTT TGA ATT ATC TGA TTG TTC GGA ATG TGG ACA GTC TAA ATT ACG TGT ACG TGA AAT TAA AGT AAC TGC AAC TGA AAG TAT TGA TTGTTC CAA
*GC% Content for gene (codon optimized): 26.6%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 1280 [NCBI]
*Protein ID (NP or XP #) or Wolbachia#: NP_878023.1
*Organism (including strain): staphylococcus aureus PC1
Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up): Staphylococcus aureus is a gram-positive bacteria grouped with Bacillus sp based on ribosomal RNA sequences. This coccus bacterium grows in both aerobic and anaerobic conditions, forming grape-like clusters. Its mainly resides in nasal membranes and skin of warm-blooded animals, causing infections ranging from mild, such as skin infections and food poisoning, to life-threatening, such as pneumonia, sepsis, osteomyelitis, and infectious endocarditis.1 The organism is highly resistant to antibiotics, including meticillin and the most potent β-lactams. Meticillin-resistant S aureus (MRSA) is the main cause of nosocomial infection worldwide and especially prominent in hospital. Beta-lactamase is involved in bacterial resistance to beta-lactam antibiotics and if a drug can inhibit this enzyme, then maybe the antiibiotics can work.
Link to TDR Targets page (if present): None
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
1280 [NCBI]
https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=RefSeq.46170.176.LFUU01000013.CDS.6906.7472.fwd#
Essentiality of this protein:
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): It is monomeric.
The essentiality is pretty high in MRSA infection because beta-lactamase is behind the resistance for beta-lactam antibiotics
Complex of proteins?: No - but it is in the group 2a of beta-lactamase proteins
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Yes, existing drugs are listed here:
http://database.idrb.cqu.edu.cn/TTD/ZFTTDDetail.asp?ID=TTDS00228
It is druggable – similar to PBP4 target and has some known drugs already
*EC#: 3.5.2.6
Link to BRENDA EC# page:
http://www.brenda-enzymes.info/enzyme.php?ecno=3.5.2.6&Suchword=&organism%5B%5D=Staphylococcus+aureus+PC1&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic - No schematic given
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Assay – penicillinase Cephaloridine as substrate
1KU - $48.50, 10KU - $297 – Sigma
25MG - $329.50
Reagents:
- 100 mM Potassium Phosphate Buffer, pH 7.0 at 25°C Sigma Prod.No. P- 5379 -100g = $22.10
- Cephaloridine, Sigma Prod.No. C-3519.) – DISCONTINUED on sigma-aldrich
- Penicillinase Enzyme Solution - 1KU = $48.50, 10KU = $297 – Sigma producer
25MG = $329.50Structure (PDB or Homology model)
-- PDB #: 1ALQ
-- Resolution = 1.8 Amps
No significant similarity between beta-lactamase and human proteins found. Blasted twice to make sure.
Current Inhibitors:
This target has a lot of compounds but none of the source organisms are staphylococcus aureus and checking the targets of staphylococcus aureus, 4 main targets are listed, and this is not one them – indicating this is a pretty unstudied target in this organism. - http://www.bindingdb.org/bind/BySourceOrganism.jsp
Boronic acids are active-site inhibitors of beta lactamases.
Expression Information (has it been expressed in bacterial cells):
Yes, its expression system is E.coli
Purification Method:
Sequential cation exchange and affinity chromatography using a phenylboronic acid-agarose affinity column to purify beta lactamase from various bacterial species.
Image of protein (PyMol with features delineated and shown separately):
Dashed lines represent hydrogen bonds
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNTKFLGKTLVASALVLTTLGTGLHSSYLGLDTNKVVKTAKAEEKMTDGQLWKKVKDSLH
DSDIILSNEYETINVTYLLSNGYSSSVSAPGNDDGGHLTQSIDFKGLKQIDLTKENVYDD
FNKKLDAKNTWNSLTEKLKGLGLLQNGQKVSIYSSDSSSPVSGKVGEGVTSGGENTLTKR
FINKITID
*length of your protein in Amino Acids: 188 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20.345 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 19940 M^-1 cm^-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
<span style="color: #626262; font-size: 12px;"> inside->outside | outside->inside 9- 31 (23) 1501 ++ | 5- 24 (20) 1108 </span>*CDS Gene Sequence (paste as text only):
TTAAAATTCCTTCATTACACTCTTGGCGGTTTCACTTATCAACTTATCATTTGGCTTATCACTTTTATTG
TCTTTATTCGTAAAAATGACTAAAACAATAGGTTCAGATTGGCCCTTAGGATAAACAAAAGCAACATCAT
TTCTAGAAGCATATGTTATTGCTTGACCACTTTTATCAGCAACCTTATAGTCTTTTGGAACACCGTCTTT
AATTAAAGTATCTCCGCTTTTATTATTTAACATTAAATCAAGTAAGAATTTTTTGTTTTCTTTGCTTAAT
TTTCCATTTGCGATAAGTTTATTTAAAGTCTTACCGAAAGCAGCAGGTGTTGAAGTATCTTTTTTGCTCT
TTGGTGAATAGTAATTTAATTCTATCTCATATCTAACTGGATTTGTTACTTTATCTCCTAGTTCTTTTAG
ACGTTGTTTAACTTTTTTGATTCCACCGATTTCTTTTATAATTTTATTGTTTGCTGTATTATCACTATAT
GTCATTGAAGCCTCAATAAGTTCTTTTAAAGTGATATCTTTTCCTACATATTTTTCTAAAATAGGAGAAT
AAGCAACTATATCATCTTTGTTAATATGTATTTTTTTATTTAACTTATTATAAGGTACTTGTTCTAACAA
AATAGCACTATTTATCGCTTTTGAAGTCGAAGCATAGGCAAATCTCTTATCTGAATTAAATTTTACTTCC
TTACCACTTTTAGTATCTAAAGCATAAACACCAATATGAGCATTATATTTTTTTTCTAAATCATTTAACT
CTTTGGCATGTGGACTGTTTGAATTACATGCACTTAAAACTAAAGCAATTGCAATTAAAAATATTAACTT
TTTCAA
http://www.ncbi.nlm.nih.gov/protein/YP_003347954.1
*GC% Content for gene: 27.78%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
TTA AAA TTC TTA CAC TAC ACA TTA GGA GGT TTC ACT TAC CAG TTA ATC ATA TGG TTG ATC ACG TTT ATA GTA TTC ATA AGA AAG AAC GAC TGA AAC AAT AGA TTCCGA TTA GCT TTG CGA ATT AAC AAA TCA AAT ATC ATT AGT CGA AGT ATA TGT TAT TGC TTG ACG ACG TTT ATT AGT AAT TTA ATA GTC TTT TGG AAT ACT GTT TTTAAC TAA TCT ATC AGT GCA TTC ATA ATT TGA CAT TGA ATT AAA TAA GAA TTT TTT GTC TTC TTC GCT TAA TTT AGT ATC TGT GAT AAG TTT ATC TGA AGT TTA ACTGAA AGT TCA CGT TGC TAA TCA ATT TTT TTC GCT TTA TGG TAA ATC GTC ATC TGA TTT TAT TTA ATA AGT AAC TGG ATC TGT TAC TTT ATT TCT TAA TTC TTC TGAACG TTA TTT AAT TTT TTC GAT TCA ACT GAT TTC TTT TAC AAC TTT ATT GTT TGT TGC ATA ATC ACA ATC TGC CAC TGA AGT TTA AAT AAA TTC TTT TGA AGT GATATC TTT TCA TAC ATC TTC TTC TGA AAT CGA CGA ATA TCT AAC TAT ATA ATT TTT GTT AAT ATG TAT TTC TTT ATA TAA TTA ATC ATT CGT TAC TTG TTT TGA CAGAAC TCT ACA ATT TAC CGT TTT TAA TCT CGT AGT ATA GGC AAA AGT TTG ATT TAA ATA AAA TTT TAC TTC TTA ACT ACT TTC TCT ATA TAA TCA ATC AAT ACA AATATG TCA ATT ATA TTC TTC TTT TGA ATT ATC TGA TTG TTC GGA ATG TGG ACA GTC TAA ATT ACG TGT ACG TGA AAT TAA AGT AAC TGC AAC TGA AAG TAT TGA TTGTTC CAA
*GC% Content for gene (codon optimized): 26.6%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**